Pluripotent genes, such as Oct4 and nanog are essential to maintain self-renewal and the ability to differentiate into all cell types in ES cells. During ES cell differentiation repression and silencing of pluripotent genes occurs concomitantly with' the the expression of early lineage determinants. We have shown that the orphan nuclear receptor GCNF (Germ Cell Nuclear Factor) plays a pivotal role in the repression of pluripotent gene expression by binding Oct4 and nanog promotor targets. GCNF"'" mES cells fail to repress pluripotent gene expression and as a consequence grow robustly in the absence of leukemia inhibitory factor (LIF). Preliminary data suggest that in addition to silencing pluripotency genes, GCNF restricts precocious expression of lineage determinants during early differentiation. The mechanism of repression involves GCNF interactions with multiple co-repressor complexes, including the nuclear receptor co-repressor (NCoR), and with multiple components of the DNA methylation machinery, including DNA methyltransferases and Methyl Binding Domain (MBD) factors, which are components of histone deacetylation complexes. Because of its pivotal role in repression of ES cell gene expression, it is critical that we better understand the molecular role of this represser in the regulation of hES cell pluripotency. The hypothesis that we will test is that GCNF establishes a transient window during differentiation of human EScells, by inhibiting early expression of lineage determinants, which permits orderly repression of pluripotent gene expression by recruiting various co-repressor complexes to target gene promoters. To test this hypothesis, we have the following aims: (1) define the role of hGCNF in repression of known target genes during hES cell differentiation, such as Oct4, nanog and Cripto. In addition, functional synergy between GCNF and p53 in the repression of Nanog expression will be investigated in collaboration with Project 3. (2) define the role of GCNF in repression of pluripotency genes and lineage markers during hES cell differentiation. A genome-wide approach to defining the regulation of gene expression during human ES cell differentiation. We will use a combination of Affymetric microarray analyses and ChlP-on-chip experiments to define the genes directly and indirectly regulated by GCNF. These profiles will be compared with those obtained for p53, and THAP11 in Projects 3 and 4. (3) define the GCNF-dependent mechanism of gene silencing during hES cell differentiation. GCNF interacting factors will be isolated from hES cells using a biochemical approach and the recruitment of candidate corepressors to pluripotency promoters will be analyzed during hES cell differentiation and correlated with temporal epigenetic changes occurring at these promoters. We will also look for specific interactions with THAP11, p53, and NANOG (with Projects 1, 3, and 4). .